Angiogenesis is critical for tumor growth and metastasis and thus targeting tumor angiogenesis is a recognized means of blocking tumor growth. Chloride Intracellular Channel (CLIC) proteins, CLIC1 and CLIC4, are newly discovered angiogenic regulators that have been implicated in tumorigenesis. This proposal will first focus on the function of Clic4, where preliminary findings show that Clic4 deficiency leads to increased tumor metastasis. We hypothesize this increased metastasis is due to dysfunctional endothelium caused by reduced vascular wall barrier functions in Clic4 mutants. We propose to implant tumor cells on mice with an endothelial specific ablation of Clic4 and monitor tumor vessels, growth and metastasis. Next, we will determine if Clic1 and Clic4, the endothelially-expressed Clic genes, are required for tumor angiogenesis. We have found that deletion of both Clic1 and Clic4 genes in mice leads to embryonic lethality due to severe vascular defects. Thus, we propose to remove Clic4 in endothelial cells using a tissue specific cre recombinase driven by the Vegfr-2 regulatory region. The endothelial specific loss of Clic4 (Clic4ECKO) will be carried out in a Clic1 null background to study combined Clic1 and Clic4 function in tumor endothelium. To address the role of Clic1 and Clic4 in tumor angiogenesis, tumors will be implanted onto the Clic1-/- ;Clic4ECKO animals and tumor growth and angiogenesis will be evaluated. When evaluating tumor vasculature, the focus will be on assessing the amount, integrity, and patency of tumor vessels using immunohistochemical analysis. The level of tumor hypoxia will be examined, as a measure of reduced tumor vascular perfusion. The effects of combined loss of Clic1/Clic4 in tumor endothelium on tumor metastasis will also be determined. We have recently discovered the CLICs function in the S1P signaling pathway, a known signaling cascade involved in endothelial cell function. This proposal will investigate if CLICs function through the S1P signaling pathway to regulate endothelial cell migration and vascular barrier function using cultured primary endothelial cells with reduced expression of either CLIC1 or CLIC4. Using vitro assays we will determine CLICs are required for S1P functions in migration, vascular permeability and endothelial junction assembly. Determining whether CLICs function in tumor angiogenesis and metastasis and elucidating the molecular mechanism that mediate their function will provide new targets for development of anti-cancer therapies that can target both tumor growth and metastasis.